Peptide selection using in vitro display method such as ribosome display (Reference Documents 1 and 2) or mRNA display (Reference Documents 3 and 4) is a leading method for searching novel functional peptides from a highly diverse peptide library. A library comprised of peptides having a cyclic structure or peptides having an N-alkylated peptide backbone in in vitro display method becomes also useful for screening of a drug candidate targeting an intracellular disease-associated molecule or a molecule having protease activity because the peptides obtain enhanced resistance against proteolysis, cell membrane permeability, and conformational rigidity.
It has so far been shown in fact that peptides macrocyclized after translation is used for in vitro peptide selection (Reference Documents 6 to 10) and in some cases, selected cyclic peptides have higher affinity for a target protein than a linear peptide corresponding thereto (Reference Documents 6, 8, and 10). With respect to N-alkylation of a peptide backbone, ribosomal synthesis of N-methyl peptide and peptoid has been developed in order to enhance membrane permeability of peptides or resistance against proteolysis (Reference Documents 11 to 16).
Further, a partially N-methylated macrocyclic peptide library constructed in a ribosomal translation system is used for selecting novel macrocyclic n-methyl peptides by using mRNA display (Reference Document 17). In the macrocyclic N-methyl peptides, both N-methylation and a cyclic structure are important for resistance against proteolysis (Reference Document 17). Excellent pharmacological properties produced by the cyclic structure and N-alkyl peptide structure show that cyclic N-alkyl amino acids (CNA) will be a useful building block for simultaneously enhancing cell permeability, resistance against proteolysis, and conformational rigidity.
Adaptability of CNA to a ribosomal translation system is important for using CNA-containing ribosomally synthesized peptides for selection. In classical in vitro nonsense suppression using a translation system with a cell extract, a ribosomal translation mechanism allows some CNAs (2, 3, and 4 in FIG. 1a) to serve as a substrate (Reference Documents 18 to 20). It has been shown that CNAs 7, 8, and 22 of FIG. 1a) charged onto tRNA by a wild type prolyl-tRNA synthetase (ProRS) are incorporated into ribosome in vivo (Reference Documents 21 to 23). These methods however fail to provide a uniform translation product with good reproducibility because they compete with termination of translation by an endogenous release factor-1 (RF1) or incorporation of natural proline. Therefore, these methods cannot be used for preparation of a peptide library including non-proteinogenic CNA for peptide selection.
Two groups have studied incorporation of non-proteinogenic CNA into ribosome by using a reconstituted cell-free translation system (Reference Documents 24 to 33). Forster, et al. have reported (Non-patent Document 1) that 3-trans-hydroxyproline (3 of FIG. 1A) chemoenzymatically charged onto tRNA is incorporated in a peptide at an efficiency equal to that of alanine or phenylalanine (Non-patent Document 1). Forster, et al. have suggested in addition that CNAs (proline and 3-trans-hydroxyproline) are likely to be incorporated into a peptide not by a linear N-alkyl amino acid (N-methyl amino acid and N-butyl amino acid) but by a translation apparatus. Foster, et al. however have actually shown incorporation of only one 3-trans-hydroxyproline into a peptide but they do not perform ribosomal translation of a peptide containing a plurality of non-proteinogenic CNA-tRNAs (Non-patent Document 2). This is presumed to result from difficulty in chemoenzymatic acylation of tRNA (Reference Documents 34 to 37).
Szostak, et al. have shown that four CNAs including thiazolidine-2-carboxylic acids (6 of FIG. 1A), thiazolidine-4-carboxylic acid (5 of FIG. 1A), and 3,4-dehydroproline (22 of FIG. 1A) serve as a substrate of ProRS and a translation apparatus (Non-patent Documents 3 and 4). The thiazolidine-4-carboxylic acid is used as a building block in mRNA display selection, showing usefulness of non-proteinogenic CNA in in vitro peptide selection (Reference Document 10). In tRNA acylation method using this ProRS catalyst, however, a plurality of different CNAs cannot be simultaneously incorporated into different codons because CNA is incorporated only into the codon representing proline. Further, some CNAs do not serve as a substrate for aminoacyl tRNA synthase (aaRS) (Reference Document 38) so that translation using CNAs is limited to the number of CNAs having good affinity for aaRS.
Thus, the study by two groups has revealed that a limited kind of CNAs is incorporated into a peptide and the number of the CNAs incorporated into the peptide is one, but due to limitation in the tRNA acylation method employed, there has been no report on comprehensive adaptability screening or incorporation of many different CNAs in a plurality of sites.